During the winter and spring of 1952-1953, Rowe and his colleagues at the National Institutes of Health (NIH) obtained and placed in tissue culture adenoids that had been surgically removed from young children in the Washington, D.C. area (Rowe et al., Proc. Soc. Exp. Biol: Med., 84, 570-573 (1953)). After periods of several weeks, many of the cultures began to show progressive degeneration characterized by destruction of epithelial cells. This cytopathic effect could be serially transmitted by filtered culture fluids to established tissue cultures of human cell lines. The cytopathic agent was called the "adenoid degenerating" (Ad) agent. The name "adenovirus" eventually became common for these agents. The discovery of many prototype strains of adenovirus, some of which caused respiratory illnesses, followed these initial discoveries (Rowe et al., supra; Dingle et al., Am. Rev. Respir. Dis., 97, 1-65 (1968); reviewed in Horwitz, "Adenoviridae and their replication," In Virology, Fields et al., eds., 2nd ed., Raven Press Ltd., New York, N.Y., pp. 1679-1721 (1990)).
Over 40 adenoviral subtypes have been isolated from humans and over 50 additional subtypes have been isolated from other mammals and birds (reviewed in Ishibashi et al., "Adenoviruses of animals," In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 497-562 (1984); Strauss, "Adenovirus infections in humans," In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 451-596 (1984)). All these subtypes belong to the family Adenoviridae, which is currently divided into two genera, namely Mastadenovirus and Aviadenovirus. All adenoviruses are morphologically and structurally similar. In humans, however, adenoviruses show diverging immunological properties and are, therefore, divided into serotypes. Two human serotypes of adenovirus, namely Ad2 and Ad5, have been studied intensively and have provided the majority of information about adenoviruses in general.
Adenoviruses are nonenveloped, regular icosahedrons, 65-80 nm in diameter, consisting of an external capsid and an internal core. The capsid is composed of 20 triangular surfaces or facets and 12 vertices (Horne et al., J. Mol. Biol., 1, 84-86 (1959)). The facets are comprised of hexons and the vertices are comprised of pentons. A fiber projects from each of the vertices. In addition to the hexons, pentons, and fibers, there are eight minor structural polypeptides, the exact positions of the majority of which are unclear. One minor polypeptide component, namely polypeptide IX, binds at positions where it can stabilize hexon-hexon contacts at what is referred to as the group-of-nine center of each facet (Furcinitti et al., EMBO, 8, 3563-3570 (1989)). The minor polypeptides VI and VIII are believed to stabilize hexon-hexon contacts between adjacent facets, and the minor polypeptide IIIA, which is known to be located in the regions of the vertices, is suggested to link the capsid and the core (Stewart et al., Cell, 67, 145-154 (1991)).
The viral core contains a linear, double-stranded DNA molecule with inverted terminal repeats (ITRs), which vary in length from 103 bp to 163 bp (Garon et al., PNAS USA 69, 2391-2394 (1972); Wolfson et al., PNAS USA, 69, 3054-3057 (1972); Arrand et al., J. Mol. Biol., 128, 577-594 (1973); Steenberg et al., Nucleic Acids Res., 4, 4371-4389 (1977); and Tooze, DNA Tumor Viruses, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory. pp. 943-1054 (1981)). The ITRs harbor origins of DNA replication (Garon et al., supra; Wolfson et al., supra; Arrand et al., supra; Steenberg et al., supra). The viral DNA is associated with four polypeptides, namely V, VII, .mu., and terminal polypeptide (TP). The 55 kd TP is covalently linked to the 5' ends of the DNA via a dCMP (Rekosh et al., Cell, 11, 283-295 (1977); Robinson et al., Virology, 56, 54-69 (1973)). The other three polypeptides are noncovalently bound to the DNA and fold it in such a way as to fit it into the small volume of the capsid. The DNA appears to be packaged into a structure similar to cellular nucleosomes as seen from nuclease digestion patterns (Corden et al., PNAS USA, 73, 401-404 (1976); Tate et al., Nucleic Acids Res., 6, 2769-2785 (1979); Mirza et al., Biochim. Biophys. Acta, 696, 76-86 (1982)).
The overall organization of the adenoviral genome is conserved among serotypes, such that specific functions are similarly positioned. The Ad2 and Ad5 genomes have been completely sequenced and sequences of selected regions of genomes from other serotypes are available.
Adenovirus begins to infect a cell by attachment of the fiber to a specific receptor on the cell membrane (Londberg-Holm et al., J. Virol., 4, 323-338 (1969); Morgan et al., J. Virol., 4, 777-796 (1969); Pastan et al., "Adenovirus entry into cells: some new observations on an old problem," In Concepts in Viral Pathogenesis, Notkins et al., eds., Springer-Verlag, New York, N.Y., pp. 141-146 (1987)). Then, the penton base binds to an adenoviral integrin receptor. The receptor-bound virus then migrates from the plasma membrane to clathrin-coated pits that form endocytic vesicles or receptosomes, where the pH drops to 5.5 (Pastan et al., Concepts in Viral Pathogenesis, Notkins and Oldstone, eds. Springer-Verlag, N.Y. pp. 141-146 (1987)). The drop in pH is believed to alter the surface configuration of the virus, resulting in receptosome rupture and release of virus into the cytoplasm of the cell. The viral DNA is partially uncoated, i.e., partially freed of associated proteins, in the cytoplasm while being transported to the nucleus.
When the virus reaches the nuclear pores, the viral DNA enters the nucleus, leaving most of the remaining protein behind in the cytoplasm (Philipson et al., J. Virol., 2, 1064-1075 (1968)). However, the viral DNA is not completely protein-free--at least a portion of the viral DNA is associated with at least four viral polypeptides, namely V, VII, TP and .mu., and is converted into a viral DNA-cell histone complex (Tate et al., Nucleic Acids Res., 6, 2769-2785 (1979)).
The cycle from cell infection to production of viral particles lasts 1-2 days and results in the production of up to 10,000 infectious particles per cell (Green et al., Virology, 13, 169-176 (1961)). The infection process of adenovirus is divided into early (E) and late (L) phases, which are separated by viral DNA replication, although some events which take place during the early phase also take place during the late phase and vice versa. Further subdivisions have been made to fully describe the temporal expression of viral genes.
During the early phase, viral mRNA, which constitutes a minor proportion of the total RNA present in the cell, is synthesized from both strands of the adenoviral DNA present in the cell nucleus. At least five regions, designated E1-4 and MLP-L1, are transcribed (Lewis et al., Cell, 7, 141-151 (1976); Sharp et al., Virology, 75, 442-456 (1976); Sharp, "Adenovirus transcription," In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 173-204 (1984)). Each region has a distinct promoter(s) and is processed to generate multiple mRNA species, and, therefore, each region may be thought of as a gene family.
The products of the early (E) regions serve regulatory roles for the expression of other viral components, are involved in the general shut-off of cellular DNA replication and protein synthesis, and are required for viral DNA replication. The intricate series of events regulating early mRNA transcription begins with expression of immediate early regions E1A, L1 and the 13.5 kd gene (reviewed in Sharp (1984), supra; Horwitz (1990), supra). Expression of the delayed early regions E1B, E2A, E2B, E3 and E4 is dependent on the E1A gene products. Three promoters, the E2 promoter at 72 map units (mu), the protein IX promoter, and the IVa promoter are enhanced by the onset of DNA replication but are not dependent on it (Wilson et al., Virology, 94, 175-184 (1979)). Their expression characterizes an intermediate phase of viral gene expression. The result of the cascade of early gene expression is the start of viral DNA replication.
Adenoviral DNA replication displaces one parental single-strand by continuous synthesis in the 5' to 3' direction from replication origins at either end of the genome (reviewed in Kelly et al., "Initiation of viral DNA replication," In Advances in Virus Research, Maramorosch et al., eds., Academic Press, Inc., San Diego, Calif., 34: 1-42 (1988); Horwitz (1990), supra; van der Vliet, "Adenovirus DNA replication in vitro," In The Eukaryotic Nucleus, Strauss et al., eds., Telford Press, Caldwell, N.J. 1: 1-29 (1990)). Three viral proteins encoded from E2 are essential for adenoviral DNA synthesis: the single-stranded DNA binding protein (DBP), the adenoviral DNA polymerase (Ad pol), and the pre-terminal protein (pTP). In addition to these, in vitro experiments have identified many host cell factors necessary for DNA synthesis.
DNA synthesis is initiated by the covalent attachment of a dCMP molecule to a serine residue of pTP. The pTP-dCMP complex then functions as the primer for Ad pol to elongate. The displaced parental single-strand can form a panhandle structure by base-pairing of the inverted terminal repeats. This terminal duplex structure is identical to the ends of the parental genome and can serve as an origin for the initiation of complementary strand synthesis.
Initiation of viral DNA replication appears to be essential for entry into the late phase. The late phase of viral infection is characterized by the production of large amounts of the viral structural polypeptides and the nonstructural proteins involved in capsid assembly. The major late promoter (MLP) becomes fully active and produces transcripts that originate at 16.5 mu and terminate near the end of the genome. Post-transcriptional processing of this long transcript gives rise to five families of late mRNA, designated L1-5 (Shaw et al., Cell, 22, 905-916 (1980)). The mechanisms which control the shift from the early to late phase and result in such a dramatic shift in transcriptional utilization are unclear. The requirement for DNA replication may be a cis-property of the DNA template, since late transcription does not occur from a superinfecting virus at a time when late transcription of the primary infecting virus is active (Thomas et al., Cell, 22, 523-533 (1980)).
Assembly of the virion is an intricate process from the first step of assembling major structural units from individual polypeptide chains (reviewed in Philipson, "Adenovirus Assembly," In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y. (1984), pp. 309-337; Horwitz (1990), supra). Hexon, penton base, and fiber assemble into trimeric homopolymer forms after synthesis in the cytoplasm. The 100 kd protein appears to function as a scaffolding protein for hexon trimerization and the resulting hexon trimer is called a hexon capsomere. The hexon capsomeres can self-assemble to form the shell of an empty capsid, and the penton base and fiber trimers can combine to form the penton when the components are inside the nucleus. The facet of the icosahedron is made up of three hexon capsomeres, which can be seen by dissociation of the capsid, but the intermediate step of formation of a group-of-nine hexons has not been observed. Several assembly intermediates have been shown from experiments with temperature-sensitive mutants. The progression of capsid assembly appears dependent on scaffolding proteins, 50 kd and 30 kd, and the naked DNA most probably enters the near-completed capsid through an opening at one of the vertices. The last step of the process involves the proteolytic trimming of the precursor polypeptides pVI, pVII, pVIII and pTP, which stabilizes the capsid structure, renders the DNA insensitive to nuclease treatment, and yields a mature virion.
Recombinant adenoviral vectors have been used in gene therapy. The use of a recombinant adenoviral vector to transfer one or more recombinant genes enables targeted delivery of the gene or genes to an organ, tissue, or cells in need of treatment, thereby overcoming the delivery problem encountered in most forms of somatic gene therapy. Furthermore, recombinant adenoviral vectors do not require host cell proliferation for expression of adenoviral proteins (Horwitz et al., In Virology, Raven Press, New York, 2, 1679-1721 (1990); and Berkner, BioTechniques, 6, 616 (1988)) and, if the diseased organ in need of treatment is the lung, has the added advantage of being normally trophic for the respiratory epithelium (Straus, In Adenoviruses, Plenum Press, New York, pp. 451-496 (1984)).
Other advantages of adenoviruses as potential vectors for human gene therapy are as follows: (i) recombination is rare; (ii) there are no known associations of human malignancies with adenoviral infections despite common human infection with adenoviruses; (iii) the adenoviral genome (which is linear, double-stranded DNA) currently can be manipulated to accommodate foreign genes ranging in size from small peptides up to 7.0-7.5 kb in length; (iv) an adenoviral vector does not insert its DNA into the chromosome of a cell, so its effect is impermanent and unlikely to interfere with the cell's normal function; (v) the adenovirus can infect non-dividing or terminally differentiated cells, such as cells in the brain and lungs; and (vi) live adenovirus, having as an essential characteristic the ability to replicate, has been safely used as a human vaccine (Horwitz, M. S. et al.; Berkner et al.; Straus et al.; Chanock et al., JAMA, 195, 151 (1966); Haj-Ahmad et al., J. Virol., 57, 267 (1986); and Ballay et al., EMBO, 4, 3861 (1985)).
Until now, adenoviral vectors used to express a foreign gene have been deficient in only a single early region (E1) that is essential for viral growth, i.e., singly functionally deficient. Only the essential region E1 or, alternatively, the nonessential region E3 has been removed for insertion of a foreign gene into the adenoviral genome. If the region removed from the adenovirus is essential for the virus to grow, a complementing system, such as a complementing cell line is necessary to compensate for the missing viral function. In other words, the complementing cell line will express the missing viral function so that the singly deficient adenovirus can grow inside the complementing cell. Currently, there are only a few cell lines that exist that will complement for essential functions missing from a singly deficient adenovirus. Examples of such cell lines include HEK-293 (Graham et al., Cold Spring Harbor Svmp. Ouant. Biol., 39, 637-650 (1975)), W162 (Weinberg et al., PNAS USA, 80, 5383-5386 (1983)), and gMDBP (Klessig et al., Mol. Cell. Biol., 4, 1354-1362 (1984); Brough et al., Virology, 190, 624-634 (1992)).
Foreign genes have been inserted into two major regions of the adenoviral genome for use as expression vectors. Insertion into the E1 region results in defective progeny that require either growth in complementary cells or the presence of an intact helper virus (Berkner et al., J. Virol., 61, 1213-1220 (1987); Davidson et al., J. Virol., 61, 122-1239 (1987); and Mansour et al., Mol. Cell Biol., 6, 2684-2694 (1986)). This region of the genome has been used most frequently for expression of foreign genes. Such E1-defective expression vector viruses usually have been grown in the HEK-293 cell line, which contains and expresses the complementing adenoviral E1 region. The inserted genes have been placed under the control of various promoters and most produce large amounts of the foreign gene product, dependent on the expression cassette. These adenoviral vectors, however, are defective in noncomplementing cell lines. In contrast, the E3 region is nonessential for virus growth in tissue culture, and replacement of this region with a foreign gene expression cassette leads to a virus that can productively grow in a noncomplementing cell line. The insertion and expression of the hepatitis B surface antigen in the E3 region with subsequent inoculation and formation of antibodies in the hamster has been reported (Morin et al., PNAS USA, 84, 4626-4630 (1987)).
The problem with singly deficient adenoviral vectors is that they limit the amount of usable space within the adenoviral genome for insertion and expression of a foreign gene. Due to similarities, or overlap, in the viral sequences contained within the singly deficient adenoviral vectors and the complementing cell lines that currently exist, recombination events can take place and create replication competent viruses within a vector stock. This event can render a stock of vector unusable for gene therapy purposes as a practical matter.
Accordingly, it is an object of the present invention to provide multiply deficient adenoviral vectors that can accommodate insertion and expression of larger pieces of foreign DNA. It is another object of the present invention to provide cell lines that complement the present inventive multiply deficient adenoviral vectors. It is also an object of the present invention to provide recombinants of multiply deficient adenoviral vectors and therapeutic methods, particularly relating to gene therapy, vaccination, and the like, involving the use of such recombinants. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.